The overall objective is to define the structure function relationships of human apolipoprotein (apo) E, especially of the isoforms apoE3 and apoE4, to provide a basis for understanding the different cardiovascular disease risks associated with these two proteins. A range of engineered apoE molecules is being used to address 3 specific aims. 1) To determine the secondary structures of human apoE3 and apoE4 in lipid free and lipid bound states using hydrogendeuterium exchange and mass spectrometry methods. The locations and stabilities of 1helical segments in the C-terminal domain of these proteins will be determined to test the hypothesis that these parameters are different in these apoE isoforms. 2) To use a range of physical biochemical methods to identify the region in the C-terminal domain of apoE3 and apoE4 responsible for their different lipid binding properties and lipoprotein binding preferences, and to elucidate the mechanistic basis for these effects. The hypothesis being tested is that the polymorphism alters the structure in the region around residues 260270 so that apoE4 binds better to lipid surfaces. 3) To use adeno-associated viral vectors to express engineered forms of human apoE3 and apoE4 in mice and assess the functional consequences for cholesterol levels and lipoprotein profiles. The hypothesis being tested is that the structure of the C-terminal region spanning residues 260270 controls the different effects that apoE3 and apoE4 have on lipoprotein levels in vivo. Overall, achievement of these 3 aims will generate novel quantitative information about the ways in which apoE structure and polymorphism affect the functional properties of the protein with respect to lipid transport. The design of apoEmimetic molecules and of ways to control the aberrant behavior of apoE4 will be facilitated by this understanding. PUBLIC HEALTH RELEVANCE: In the human population, apoE is expressed in 3 common forms that function differently in regulating lipid transport in vivo. As a result, these mutations in the apoE molecule can increase the risk for development of both cardiovascular disease and Alzheimer's disease. This project will provide more insights into the molecular mechanisms underlying these pathological effects.